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Subunit interfaces contribute differently to activation and allosteric modulation of neuronal nicotinic acetylcholine receptors.

ABSTRACT: Neuronal nicotinic acetylcholine receptors (nAChRs) are widely distributed in the nervous system and are implicated in many normal and pathological processes. The structural determinants of allostery in nAChRs are not well understood. One class of nAChR allosteric modulators, including the small molecule morantel (Mor), acts from a site that is structurally homologous to the canonical agonist site but exists in the ?(+)/?(-) subunit interface. We hypothesized that all nAChR subunits move with respect to each other during channel activation and allosteric modulation. We therefore studied five pairs of residues predicted to span the interfaces of ?3?2 receptors, one at the agonist interface and four at the modulator interface. Substituting cysteines in these positions, we used disulfide trapping to perturb receptor function. The pair ?3Y168-?2D190, involving the C loop region of the ?2 subunit, mediates modulation and agonist activation, because evoked currents were reduced up to 50% following oxidation (H2O2) treatment. The pair ?3S125-?2Q39, below the canonical site, is also involved in channel activation, in accord with previous studies of the muscle-type receptor; however, the pair is differentially sensitive to ACh activation and Mor modulation (currents decreased 60% and 80%, respectively). The pairs ?3Q37-?2A127 and ?3E173-?2R46, both in the non-canonical interface, showed increased currents following oxidation, suggesting that subunit movements are not symmetrical. Together, our results from disulfide trapping and further mutation analysis indicate that subunit interface movement is important for allosteric modulation of nAChRs, but that the two types of interfaces contribute unequally to receptor activation.

SUBMITTER: Short CA

PROVIDER: S-EPMC4332533 | biostudies-literature | 2015 Apr

REPOSITORIES: biostudies-literature

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Subunit interfaces contribute differently to activation and allosteric modulation of neuronal nicotinic acetylcholine receptors.

Short Caitlin A CA Cao Angela T AT Wingfield Molly A MA Doers Matthew E ME Jobe Emily M EM Wang Nan N Levandoski Mark M MM

Neuropharmacology 20141205

Neuronal nicotinic acetylcholine receptors (nAChRs) are widely distributed in the nervous system and are implicated in many normal and pathological processes. The structural determinants of allostery in nAChRs are not well understood. One class of nAChR allosteric modulators, including the small molecule morantel (Mor), acts from a site that is structurally homologous to the canonical agonist site but exists in the β(+)/α(-) subunit interface. We hypothesized that all nAChR subunits move with re ...[more]

PMID: 25486620

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Project description:Acetylcholine-based neurotransmission is regulated by cationic, ligand-gated ion channels called nicotinic acetylcholine receptors (nAChRs). These receptors have been linked to numerous neurological diseases and disorders such as Alzheimer's disease, Parkinson's disease, and nicotine addiction. Recently, a class of compounds has been discovered that antagonize nAChR function in an allosteric fashion. Models of human ?4?2 and ?3?4 nicotinic acetylcholine receptor (nAChR) extracellular domains have been developed to computationally explore the binding of these compounds, including the dynamics and free energy changes associated with ligand binding. Through a blind docking study to multiple receptor conformations, the models were used to determine a putative binding mode for the negative allosteric modulators. This mode, in close proximity to the agonist binding site, is presented in addition to a hypothetical mode of antagonism that involves obstruction of C loop closure. Molecular dynamics simulations and MM-PBSA free energy of binding calculations were used as computational validation of the predicted binding mode, while functional assays on wild-type and mutated receptors provided experimental support. Based on the proposed binding mode, two residues on the ?2 subunit were independently mutated to the corresponding residues found on the ?4 subunit. The T58K mutation resulted in an eight-fold decrease in the potency of KAB-18, a compound that exhibits preferential antagonism for human ?4?2 over ?3?4 nAChRs, while the F118L mutation resulted in a loss of inhibitory activity for KAB-18 at concentrations up to 100 µM. These results demonstrate the selectivity of KAB-18 for human ?4?2 nAChRs and validate the methods used for identifying the nAChR modulator binding site. Exploitation of this site may lead to the development of more potent and subtype-selective nAChR antagonists which may be used in the treatment of a number of neurological diseases and disorders.

Dataset Information

Subunit interfaces contribute differently to activation and allosteric modulation of neuronal nicotinic acetylcholine receptors.

Publications

Subunit interfaces contribute differently to activation and allosteric modulation of neuronal nicotinic acetylcholine receptors.

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